Ion channels in volume regulation of clonal kidney cells

Objectives: Clonal kidney cells (Vero cells) are extensively utilized in the manufacture of biological preparations for disease diagnostics and therapeutics and also in preparation of vaccines. In all cells, regulation of volume is an essential function coupled to a variety of physiological processes and is a topic of interest. The objective here was to investigate involvement of ion channels in the process of volume regulation of Vero cells. Methods: Involvement of ion channels in cell volume regulation was studied using video‐microscopy and flow cytometry. Pharmacologically unaltered cells of different sizes, which are presumably at different phases of the cell cycle, were used. Results: Ion transport inhibitors altered all phases of regulatory volume decrease (RVD) of Vero cells, rate of initial cell swelling, V_max and volume recovery. Effects were dependent on type of inhibitor and on cell size (cell cycle phase). Participation of aquaporins in RVD was suggested. Inhibitors decelerated growth, arresting Vero cells at the G₀/G₁ phase boundary. Electrophysiological study confirmed presence of volume‐activated Cl^? channels and K⁺ channels in plasmatic membranes of the cells. Conclusion: Vero cells of all sizes maintained the ability to recover from osmotic swelling. Activity of ion channels was one of the key factors that controlled volume regulation and proliferation of the cells.     

Hiv infection of human gastrointestinal submucosal cells: An in vitro model that mimics kaposi’s sarcoma

Summary Infection of human gastrointestinal submucosal mesenchymal cells with HIV-1 led to cell populations with abnormal growth properties, increased synthesis of endothelial cell and angioblast markers, and release of angiogenic factors. This system may be the first in vitro model for HIV-induced Kaposi’s sarcoma. Research partially supported by NIH grant DK40625.     

Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin

Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [³H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10⁻⁹ M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto; and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow.     

Placental-derived mitogenic factor for human fetal adrenocortical cell cultures

Summary The human fetal adrenal cortex is one of the largest fetal organs and synthesizes precursors for placental estrogen production as part of the feto-placental unit. The factors controlling the rapid growth of the human fetal adrenal cortex during the second and third trimesters are not known. Placental regulation of the growth of human fetal adrenocortical cell cultures from second trimester fetuses was studied. A placental-derived mitogenic factor (PDMF) was detected in tissue homogenates of 14 to 22 week human placentas and stimulated adrenocortical cell number and [³H]thymidine incorporation into DNA 5–8 fold. PDMF has been partially purified by ammonium sulfate precipitation and anion exchange chromatography. PDMF is a heat sensitive protein with disulfide bonds required for activity. The growth stimulation by PDMF was significantly greater than that for basic or acidic fibroblast growth factor by 25–50% and epidermal growth factor by 3–4 fold. The placental hormones, progesterone, estriol, estradiol, placental lactogen and chorionic gonadotropin, either alone or in combination did not stimulate fetal adrenocortical cell growth, except for a 41% cell number increase by progesterone. Platelet-derived growth factor and insulin-like growth factors I and II were not mitogenic for these cells. These results show that the placenta contains a potent growth factor for human fetal adrenocortical cell cultures. This implies a direct role for the placenta in control of this fetal organ’s growth, which would make the human feto-placental unit a bi-directional relationship. This research was supported by Grants HD15882 and HD21798 from the National Institutes of Health, Bethesda, MD. This work was presented in part at the 68th Annual Meeting of The Endocrine Society, Anaheim, CA, June 1986. Editor’s Statement This report describes a potentially new relationship between placenta and the regulation of growth of fetal adrenalcortical cells. Since these cells produce several of the essential hormones influencing fetal development, characterization of this factor(s) could provide important insights into the process of differentiation. David A. Sirbasku     

Cell Cycle Regulation by Light in Prochlorococcus Strains

The effect of light on the synchronization of cell cycling was investigated in several strains of the oceanic photosynthetic prokaryote Prochlorococcus using flow cytometry. When exposed to a light-dark (L-D) cycle with an irradiance of 25 μmol of quanta · m⁻² s⁻¹, the low-light-adapted strain SS 120 appeared to be better synchronized than the high-light-adapted strain PCC 9511. Submitting L-D-entrained populations to shifts (advances or delays) in the timing of the “light on” signal translated to corresponding shifts in the initiation of the S phase, suggesting that this signal is a key parameter for the synchronization of population cell cycles. Cultures that were shifted from an L-D cycle to continuous irradiance showed persistent diel oscillations of flow-cytometric signals (light scatter and chlorophyll fluorescence) but with significantly reduced amplitudes and a phase shift. Complete darkness arrested most of the cells in the G₁ phase of the cell cycle, indicating that light is required to trigger the initiation of DNA replication and cell division. However, some cells also arrested in the S phase, suggesting that cell cycle controls in Prochlorococcus spp. are not as strict as in marine Synechococcus spp. Shifting Prochlorococcus cells from low to high irradiance translated quasi-instantaneously into an increase of cells in both the S and G₂ phases of the cell cycle and then into faster growth, whereas the inverse shift induced rapid slowing of the population growth rate. These data suggest a close coupling between irradiance levels and cell cycling in Prochlorococcus spp.     

Clonal derivation of a rat muscle cell strain that forms contraction-competent myotubes

Summary A muscle cell strain capable of forming contracting myotubes was isolated from an established rat embryo cell line. The myogenic cells, termed rat myoblast omega or RMo cells, have a diploid complement of chromosomes (n=42). In the presence of mitogen-containing growth medium, RMo cells proliferated with a cell generation time of about 12 hours. In mitogen-depleted medium, RMo cells withdrew from the cell cycle and formed myotubes that spontaneously contracted. Differentiated RMo cells produced creatine kinase isozymes in a ratio characteristic of skeletal muscle cells. RMo cells were easy to cultivate. Cells proliferated and differentiated equally well on gelatin-coated or noncoated culture dishes, at clonal or mass culture densities, and in all basal media tested. In most experiments, growth medium consisted of horse serum-containing medium supplemented with either chicken embryo extract or FGF activity; cells proliferated equally well in medium containing unsupplemented calf serum. RMo cells differentiated if growth medium was not replenished regularly. Alternatively, differentiation was induceable by incubation in mitogen-depleted medium consisting of basal medium supplemented either with 10⁻⁶ M insulin, 0.5% serum, or 50% conditioned growth medium. RMo cells were competently transformed with cloned exogenous genes. Because it forms functional myofibrils, the RMo cell line constitutes a useful model system for studying the cell biology and biochemistry of proteins involved in contractile apparatus assembly and muscle disease. This work was supported by NIH research grant GM34432 and Research Career Development Award AG00334. Editor's Statement This report documents the characterization of a differentiating rat cell line that does not show the karyotypic shift toward polyploidy usually observed in rodent cell lines. Investigators already are finding this line valuable in studies of regulation of growth and differentiation.     

Maintenance of dihydrofolate reductase enzyme after disappearance of DHFR mRNA during muscle cell differentiation

Summary Terminally differentiating mouse muscle cells were used to examine the relationship between mytogenic withdrawal from the cell cycle and the levels of dihydrofolate reductase (DHFR) mRNA and DHFR activity. Differentiation was induced by removal of fibroblast growth factor activity from the medium. DHFR mRNA was measured by a RNase protection assay. DHFR activity was measured by a spectrophotometric assay and by a [³H]methotrexate binding assay. Proliferative myoblasts contained four DHFR mRNA molecules and 1.8×10⁵ DHFR enzyme molecules. By 12.5 h after induction, when [³H]thymidine labeling indices showed all cells had withdrawn from the cell cycle, DHFR mRNA levels had declined to 0.7 copies per cell. In contrast, myogenic withdrawal did not result in reduced DHFR activity. Qualitatively similar results, i.e. down-regulation of mRNA and constitutive expression of activity, were observed in a methotrexate-selected muscle cell line with >50-fold amplification of the DHFR gene. Enzyme synthesis rate and stability measurements indicated that persistence of DHFR activity in postreplicative cells was due to a long enzyme lifetime rather than to continued synthesis from residual normal DHFR mRNA or an alternative mRNA species not detected by the RNase protection assay. Unlike DHFR, thymidine kinase (TK) activity disappeared rapidly as muscle cells differentiated. Both DHFR mRNA and TK mRNA are expressed in a replication-dependent manner; however, the enzymes encoded by these messages are subject to different fates in postreplicative cells. This work was supported by National Institutes of Health (Bethesda, MD) research grant GM34432, NIH Research Career Development Award AG00334, and a grant from the Medical Research Foundation of Oregon to G. F. M. E. E. S. was supported in part by Predoctoral Training Grant GM07774-08 from the Department of Health and Human Services, Washington, DC. and a N. L. Tartar Research Fellowship.     

Long-term culture of human endothelial cells

Summary Human umbilical vein endothelial cells can be grown in vitro for 28 passages (CPDL 58) in Medium 199 supplemented with newborn bovine serum and a partially purified growth factor derived from bovine brain. Newborn bovine serum is superior to fetal bovine serum for the proliferation of human umbilical vein endothelial cells seeded at low density in the presence of the growth factor. The endothelial cells, which can be passaged every 7 to 10 d at a 1-to-5 split ratio, retain their morphological and biochemical characteristics. The proliferation of cells seeded at low density (10³/cm²) is proportional to the concentration of the growth factor present in the medium. The growth factor, which has an isoelectric point between 5.0 and 5.5, can support cell proliferation at reduced serum concentrations; half-maximal growth is achieved in medium containing the growth factor and 3% serum. The brain endothelial cell growth factor does not stimulate DNA synthesis significantly in cultures of human skin fibroblasts. This research was supported by grants from the U.S. Public Health Service (AG 01732, HL 16387, and HL 07080), the Cystic Fibrosis Foundation, and the New York and American Heart Associations. Victor B. Hatcher is an Established Fellow of the New York Heart Association and a recipient of the Ann Weinberg Cystic Fibrosis Research Scholarship Award.     

Cell cycle kinetics of insect cell cultures compared to mammalian cell cultures

Cell cycle kinetics of lepidopteran cell lines Sf9 (Spodoptera frugiperda) and IZDMb0503 (Mamestra brassicae) were investigated and compared to mammalian cell cycle distributions. The resting phase (G₀) of mammalian cells is characterized by a 2c-DNA content whereas G₀-phase of insect cell lines is characterized by a 4c-DNA content. Flow cytometric data in combination with growth curves of partially synchronized and asynchronously growing cells proved the existence of this phenomenon. Kinetics of cells labeled by the thymidine analog on 5-bromo-2′-deoxyuridine supported these results, which now render the possibility of applying cell cycle analysis in fermentation technology of insect cells.     

Comparative endocrinology-paracrinology-autocrinology of human adult large vessel endothelial and smooth muscle cells

Summary Endothelial and smooth muscle cells were isolated from human adult large blood vessels to compare their proliferative response to hormones and growth factors. Neural extracts and the medium from differentiated hepatoma cells were used as concentrated sources of required hormones and growth factors that supported both cell types. Active hormones and growth factors were identified from the neural extracts and hepatoma medium by substitution or direct isolation and biochemical characterization. Epidermal growth factor, lipoproteins, and heparin-binding growth factors elicited growth-stimulatory effects on both endothelial and smooth muscle cells. Both types of human vascular cells displayed 7600 to 8600 specific heparin-binding growth factor receptors per cell with a similar apparent dissociation constant (Kd) of 200 to 250 pM. Heparin modified the response of both endothelial and smooth muscle cells to heparin-binding growth factors dependent on the type of heparin-binding growth factor and amount of heparinlike material present. In addition, heparin exerted a growth factor-independent inhibition of smooth muscle cell proliferation. Platelet-derived growth factor, insulinlike growth factors, and glucocorticoid specifically supported proliferation of smooth muscle cells with no apparent effect on endothelial cell proliferation. Growth-factorlike proteinase inhibitors had an impact specifically on endothelial cell proliferation. Transforming growth factor beta was a specific inhibitor of endothelial cells, but had a positive effect on smooth muscle cell proliferation. The results provide a framework for differential control of the two vascular cell types at normal and atherosclerotic blood vessel sites by the balance among positive and negative effectors of endocrine, paracrine and autocrine origin. This research was supported by NIH grants CA37589, HL33847, and AM35310 from the National Institutes of Health, Bethesda, MD; grant 1718 from the Council for Tobacco Research; and a grant from RJR/Nabisco, Inc.     

Stimulation of human vascular endothelial cell growth by a platelet-derived growth factor and thrombin

Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet-derived growth factor (PDGF) and the protease thrombin. When 10⁴ HuE cells are seeded as a dense island (2-mm diameter) in the center of a 16-mm tissue culture well in medium containing 20% human serum derived from platelet-poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 × 10⁴. When human thrombin (1 μg/ml) is added along with the PDGF, the cell number rises to 9.2 × 10⁴. Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet-derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.     

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell′s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry¹ offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-β1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture⁵. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.     

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells resuts in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated ⁴⁵Ca⁺², while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression. © 1995 Wiley-Liss, Inc.     

Integrin Signaling at the M/G1 Transition Induces Expression of Cyclin E

The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21^cip1/Waf1 and p27^Kip1 is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment.     

Periodic oscillations in leukopoiesis models with two delays

The term leukopoiesis describes processes leading to the production and regulation of white blood cells. It is based on stem cells differentiation and may exhibit abnormalities resulting in severe diseases, such as cyclical neutropenia and leukemias. We consider a nonlinear system of two equations, describing the evolution of a stem cell population and the resulting white blood cell population. Two delays appear in this model to describe the cell cycle duration of the stem cell population and the time required to produce white blood cells. We establish sufficient conditions for the asymptotic stability of the unique nontrivial positive steady state of the model by analysing roots of a second degree exponential polynomial characteristic equation with delay-dependent coefficients. We also prove the existence of a Hopf bifurcation which leads to periodic solutions. Numerical simulations of the model with parameter values reported in the literature demonstrate that periodic oscillations (with short and long periods) agree with observations of cyclical neutropenia in patients.     

Biogenesis of plasma lipoproteins in rat hepatoma McA-RH7777: Importance of diffusion-mediated events during cell growth

Summary Cultured McA-RH7777 rat hepatoma cells actively synthesize and secrete plasma lipoproteins. However, synthesis of [¹⁴C]triglyceride declines monotonically throughout the early growth period and remains low in postconfluent cultures; and net secretion of [¹⁴C]triglyceride is 10-fold more efficient in logarithmically growing cultures than in postconfluent cultures. Secretion of apolipoproteins associated with very low density and low density lipoproteins is selectively reduced in postconfluent cultures. The temporal reductions in [¹⁴C]triglyceride production are related more strongly to increasing cell concentration (cells/cm³ medium) than to increasing cell density (cells/cm² growth surface). We have allowed cells to grow either retained within small circular corrals or unrestricted in culture dishes. When seeded at equal density (10⁴ cells/cm²) but at one-fifth the cell concentration, corralled cells synthesize twice as much [¹⁴C]triglyceride per cell after 2 and 4 d, and are 10 times as efficient in [¹⁴C]triglyceride secretion by 6 d of growth, as noncorralled cells. When seeded at equal cell concentration (10⁵ cells/dish) but at 5 times the cell density, corralled cells are only 20% less efficient at [¹⁴C]triglyceride synthesis and secretion than noncorralled cells. Conditioned medium depresses synthesis and secretion efficiency of [¹⁴C]triglyceride. Orotic acid exposure also inhibits synthesis of [¹⁴C]triglyceride and secretion of certain [³⁵S]apolipoproteins in early cultures, but it has no significant effect on late cultures. We conclude that diffusion-mediated events are important regulators of triglyceride and apolipoprotein production in growing rat hepatoma cells, but that events associated with formation of cell-to-cell contacts play a minor role in regulation of plasma lipoprotein biogenesis. This research was supported by grants to R. H. from the American Heart Association (85-805), from NHLBI (15062, Specialized Center of Research in Atherosclerosis), and from the Louis Block Fund. S. T. was supported as an American Heart Association Medical Student Research Fellow. S. T. and H. S. are recipients of predoctoral training grants from the National Institutes of Health, Bethesda, MD (HL 723711, HD 07009). This work has been presented elsewhere in preliminary form (14, 48, 49).     

Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA,and Skp2

The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3^AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3^AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27^Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3^AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3^AR cells partially blocked accumulation of p27^Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.